This application is a national stage filing under 35 U.S.C. xc2xa7371 of international application no. PCT/EP99/00930, filed on Feb. 12, 1999.
The present invention relates to calcium salts of lipopeptide antibiotics, a process for their preparation, and their use.
EP 0 629 636 A1 discloses lipopeptide antibiotics of the formula I 
in which R1 is an OH or NH2 group and R2 is a fatty acid radical (Rxe2x80x94C(O)xe2x80x94).
These lipopeptide antibiotics can be divided into two groups which differ with respect to their exocyclic amino acid: the lipopeptide antibiotics of the amphomycin type are characterized by the exocyclic amino acid aspartic acid (Asp, where R1 in formula I is an OH group) (R. C. Strong et al., Antimicrobial Agents and Chemotherapy 1970, 42-45; M. Bodanszy et al. J. Am. Chem. Soc. 95, 2352-2357 (1973)), while the lipopeptide antibiotics of the asparagine type are distinguished by the exocyclic amino acid asparagine (Asn, where R1 in formula I is an NH2 group). The lipopeptide antibiotics of the amphomycin type and of the asparagine type differ from each other by substitution on the xcex1-amino group of the exocyclic amino acid (Asp or Asn) having different fatty acid radicals (R2 in formula 1).
Furthermore, EP 0 688 789 A1 (U.S. Pat. No. 5,629,288) discloses derivatives of the lipopeptide antibiotics of the amphomycin type and of the asparagine type and their pharmaceutically tolerable salts. As pharmaceutically tolerable salts of the lipopeptide antibiotics of the formula I, EP 0 688 789 A1 (U.S. Pat. No. 5,629,288) discloses salts with inorganic and organic acids, e.g. hydrochloric acid, sulfuric acid, acetic acid, citric acid, p-toluenesulfonic acid, with inorganic and organic bases such as NaOH, KOH, Mg(OH)2, diethanolamine, ethylenediamine or with amino acids such as arginine, lysine and glutamic acid.
The calcium salt of amphomycin is furthermore known (Kirk-Othmer, Encyclopedia of Chemical Technology, fourth edition, Volume 3, Antibiotics to Batteries, John Wiley and Sons, page 284). It is only sparingly soluble in water and because of the toxicity of amphomycin as a result of its hemolytic activity on systemic use was used exclusively in antibiotic ointments for local application.
The lipopeptide antibiotics of the asparagine type (R1 in formula I is an NH2 group) and their preparation have been described for the first time in EP 0 629 636 A1. The preparation process proposed there, the fermentation of Actinoplanes sp., preferably Actinoplanes friulensis (deposited on Jun. 18, 1990 in accordance with the rules of the Budapest Convention under the Deposit No. DSM 7358 in the Deutschen Sammlung von Mikroorganismen und Zelikulturen GmbH, DSMZ, Mascheroder Weg 1b, D-38124 Brunswick), leads, however, to a mixture of a large number of the structurally closely related lipopeptides of the amphomycin type and of the asparagine type, which have very different properties, in particular different biological actions, such as, for example, their antibacterial action, toxicity as a result of their hemolytic action, and also different physicochemical properties, such as, for example, their solubility and stability, but can only be separated from the culture medium with difficulty. It would therefore be a great advantage to have a fermentation process which essentially leads to the production of preferably only one of the many possible lipopeptide components.
It is an object of the present invention to make available a salt of the lipopeptide antibiotics of the asparagine type which is distinguished by a relatively high stability and good antibacterial activity and can be administered systemically (parenterally) as a result of its good water solubility and as a result of its low toxicity, in particular on account of a low hemolytic activity.
It is further an object of the present invention to make available a process for the preparation of a salt of the lipopeptide antibiotics of the asparagine type and in particular an improved process for the fermentative preparation of its acid precursors, in which lipopeptide antibiotics of the asparagine type are preferably produced.
Finally, it is an object of the present invention to make available a pharmaceutical which contains a salt of the lipopeptide antibiotics of the asparagine type having the desired advantageous properties.
It has now been found in the case of the lipopeptide antibiotics of the asparagine type that the various salts of the same lipopeptide (or the same corresponding acid) can have very different properties. For example, the sodium salts as a rule have a very good antibacterial activity and are readily soluble in water. However, these can only be kept for a limited period, in particular at elevated temperatures. Since in the case of medicaments and other commercial products stability is of great importance, e.g. for the handling of the goods, stable salt forms of the lipopeptide antibiotics are necessary.
Since the lipopeptide antibiotics of the asparagine type are amphoteric compounds having an isoelectric point in the acidic range, neutral salts can be prepared with numerous bases. Possible cations are monovalent and polyvalent ions, such as, for example, alkali metal, alkaline earth metal and other metal ions, but also salts with ammonia or with organic bases, such as amines. Examples of the lafter are lysine and lysyllysine salts, which are very highly tolerable and have full activity.
Surprisingly, it has now been found that unlike the calcium salts of the lipopeptide antibiotics of the amphomycin type (in particular amphomycin), the calcium salts of the lipopeptide antibiotics of the asparagine type are not only active and tolerable, but also readily soluble in water and particularly stable, unlike the corresponding sodium salts.
Accordingly, the object set above is achieved by a calcium salt of the compound of the formula II 
in which R1 is a straight-chain or branched, saturated or unsaturated aliphatic acyl radical having 8 to 22 carbon atoms, which can optionally be interrupted by one or more phenyl or cycloalkyl groups or linked to such groups and can furthermore be optionally interrupted by oxygen.
Preferably, R1 in formula II is an acyl radical interrupted by a phenyl or cycloalkyl group or linked to such a group, for example 
where n is an integer from 0 to 20.
Furthermore preferred is a calcium salt of the compound of the formula II which is distinguished in that R1 is an acyl radical interrupted by a phenyl or cycloalkyl group and by oxygen, preferably wherein R1 is 
where n is an integer from 0 to 20.
Particularly preferred is a calcium salt of the compound of the formula II which is distinguished in that R1 is a straight-chain or branched, saturated or unsaturated aliphatic acyl radical having 12 to 15 carbon atoms, where R1 in formula II preferably is a fatty acid radical of the formula shown below: 
The calcium salt of the compound of the formula II can be present in two forms, as a dicalcium salt or as a monocalcium salt.
Depending on the number of anions and the fatty acid substituent (R1 in formula II), the dicalcium salt can be described in greater detail
(i) for example in the case of a saturated fatty acid (R1 in formula II) by the empirical formula
C46+nH68+2nN14O19Ca2X2,xe2x80x83xe2x80x83(ia)
C46+nH69+2nN14O19Ca2X3 orxe2x80x83xe2x80x83(ib)
C46+nH70+2nN14O19Ca2X4,xe2x80x83xe2x80x83(ic)
where in the empirical formula n is an integer from 7 to 21 and X is an anion, or
(ii) for example in the case of a monounsaturated fatty acid (R1 in formula II) by the empirical formula
C46+nH66+2nN14O19Ca2X2,xe2x80x83xe2x80x83(iia)
C46+nH67+2nN14O19Ca2X3 orxe2x80x83xe2x80x83(iib)
C46+nH68+2nN14O19Ca2X4,xe2x80x83xe2x80x83(iic)
where in the empirical formula n is an integer from 7 to 21 and X is an anion.
For example, the abovementioned, preferred calcium salts of the compound of the formula II(3c) and (3d) can be described in greater detail by the following empirical formulae if a dicalcium salt is present, where in the empirical formulae X is an anion:
C59H92N14O19Ca2X2,xe2x80x83xe2x80x83(3c, 3d/iia)
C59H93N14O19Ca2X3 orxe2x80x83xe2x80x83(3c, 3d/iib)
C59H94N14O19Ca2X4.xe2x80x83xe2x80x83(3c, 3d/iic)
Preferably, in all abovementioned empirical formulae, the anion X is a halide anion, Clxe2x88x92, Brxe2x88x92 or Ixe2x88x92, particularly preferably Clxe2x88x92.
Depending on the fatty acid substituent (R1 in formula II), the monocalcium salt can furthermore be characterized in greater detail for example in the case of a saturated fatty acid (R1 in formula II) by the empirical formula
C46+nH68+2nN14O19Caxe2x80x83xe2x80x83(iii)
or, for example, in the case of a monounsaturated fatty acid (R1 in formula II) by the empirical formula
C46+nH66+2nN14O19Ca,xe2x80x83xe2x80x83(iv)
where n in both empirical formulae is an integer from 7 to 21.
For example, the abovementioned, preferred calcium salts of the compound of the formula II (3c) and (3d) can be described in greater detail by the following empirical formula if a monocalcium salt is present:
C59H92N14O19Ca.xe2x80x83xe2x80x83(3c, 3d/iv)
In the abovementioned, preferred calcium salts of the compound of the formula II (3c) and (3d), in contrast, the corresponding acid, namely the corresponding compound of the formula II, for example has the empirical formula C59H94N14O19.
The acid A1437-D mentioned below, corresponding to the abovementioned, preferred calcium salt (3d) (the corresponding compound of the formula II) has the structure shown below (FIG. 1): 
FIG. 1: Structural formula of A1437-D (acid form of 3d)
Table 1 lists the assignment of the 1H and 13C-NMR signals as exemplified by the A1437-D sodium salt. The amino acid designations are abbreviated according to the international conventions, Dab=2,3-diaminobutyric acid, Me-Asp=xcex2-methylaspartic acid, Pip=pipecolic acid, FA=fatty acid. The amino acids preferably have the following configuration: Pip-3: D; Me-Asp4: L-threo; Asp-5, -7: L; Dab-2: L-threo; Dab-9: D-erythro; Val-10: L; Pro-1 1: L; Asn-1: L.
It was then possible to observe that, for example, in the change from the A-1437-D sodium salt to the calcium salt (3d), the physicochemical properties of the antibiotic fundamentally change. Thus the specific rotation [xcex1] of the sodium salt at the wavelength of the sodium D line and at 20xc2x0 C. rises from +6 to over +52xc2x0 if a soluble calcium salt, such as, for example, CaCl2, is added to the aqueous solution. It seems reasonable to assume from this unreasonable behavior that the molecule undergoes a significant change in its conformation. This conformational change is also supported by the low conductivity of the A-1437-D calcium salt (3d/iv), which is markedly lower than would be expected for an ionic compound.
There are several possibilities for the preparation of calcium salts of the compound of the formula II. On the one hand, the restricted solubility of the calcium salts in selected solutions can be made usable. While the Na+ or the NH4+ salts are very readily soluble in water or in methanol and are soluble in higher alcohols and other polar organic solvents, solubilities of the corresponding calcium salts in nonaqueous solvents are markedly reduced.
Accordingly, the process for the preparation of the calcium salt of the compound of the formula II described above is distinguished in that a sodium or ammonium salt of the compound of the formula II is dissolved in a suitable organic solvent, a calcium salt dissolved in ethanol is added to this solution and the calcium salt of the compound of the formula II is isolated as a precipitate. Preferably, the suitable organic solvent is ethanol. The calcium salt to be added dissolved in ethanol is preferably a calcium halide, CaCl2, CaBr2, CaI2 or the corresponding hydrates. In this process, the dicalcium salts of the compound of the formula II preferably result.
For the preparation, for example, of the calcium salts (3d/iia) having the empirical formula C59H92N14O19Ca2X2, (3d/iib) having the empirical formula C59H93N14O19Ca2X3 or (3d/iic) having the empirical formula C59H94N14O19Ca2X4, a procedure can therefore be used in which the sodium or ammonium salt of A1437D (the acid corresponding to (3d)) is dissolved in a suitable organic solvent, such as, for example, ethanol, and a calcium salt dissolved in ethanol is added to this solution. The A1437D calcium salt which is poorly soluble in the organic solvent is deposited here in the form of a precipitate. Calcium salts soluble in ethanol and suitable for the precipitation are, for example, CaCl2, CaBr2, CaI2 and their hydrates. This precipitation process results in salts which, apart from the calcium cation, can additionally contain anions of the precipitation salt, for example the halides Clxe2x88x92, Brxe2x88x92 and Ixe2x88x92. The empirical formulae of the precipitated calcium salts can, for example, read: C59H92N14O19Ca2halide2, such as,for example, C59H92N14O19Ca2Cl2 or C59H92N14O19Ca2I2 or C59H93N14O19Ca2halide3 such as, for example, C59H93N14O19Ca2Br3 or C59H93N14O19Ca2Cl3; depending on the precipitation conditions, however, other mixed salts can also be formed for which, inter alia, the composition C59H94N14O19Ca2halide4 is found. These mixed salts (A 1437 calcium mixed salts) are soluble in water and are therefore suitable for the treatment of bacterial infections or for preservation or alternatively for growth promotion in animal breeding, but they can also be used as intermediates for the preparation of other salts.
Crystallization is a further possibility of obtaining or of purifying calcium salts of the compound of the formula II. Here, the property of the antibiotic calcium salt of dissolving in pure water, in dimethyl sulfoxide, in pure methanol and in other polar solvents is used.
Accordingly, the process for the preparation of a calcium salt, preferably of the monocalcium salt, of the compound of the formula II is distinguished in that a dicalcium salt of the compound of the formula II, which, for example, can be obtained by the method described above, is dissolved in a polar solvent, then the solution obtained is treated with a less polar solvent or a mixture of less polar solvents and the calcium salt, preferably the monocalcium salt, is isolated as a precipitate.
A further process for the preparation of a calcium salt, preferably of the monocalcium salt, of the compound of the formula II is distinguished in that a sodium or ammonium salt of the compound of the formula II is dissolved in a polar solvent, a calcium salt dissolved in the same polar solvent is added to this solution, then the solution obtained is treated with a less polar solvent or a less polar solvent mixture and the calcium salt, preferably the monocalcium salt, is isolated as a precipitate.
Preferably, in the two process alternatives, the sodium or ammonium salt or the dicalcium salt of the compound of the formula II is dissolved in a polar solvent which is selected from the group consisting of water, dimethyl sulfoxide and methanol.
Preferably, the less polar solvent with which the solution obtained is treated is selected from the group consisting of alcohols, acetone and acetonitrile.
Preferably, the sodium or ammonium salt or the dicalcium salt of the compound of the formula II is dissolved in water, and the less polar solvent is methanol.
Advantageously, a mixture of methanol and butanol is added in both the process variants as a less polar solvent mixture.
Alternatively, the calcium salt, preferably the monocalcium salt, of the compound of the formula II can be prepared by dissolving a sodium or ammonium salt of the compound of the formula II in methanol, adding a calcium salt dissolved in the same solvent to this solution, then treating the solution obtained with water or a mixture of water and butanol and isolating the calcium salt, preferably the monocalcium salt, as a precipitate.
Preferably, in all these process variants the dissolved calcium salt preferably added is a calcium halide which is selected from the group consisting of CaCl2, CaBr2, CaI2 and their hydrates.
For example, the A1437-D dicalcium salts can be dissolved in concentrated form in a highly dissolving solvent and then treated with an agent which is miscible but dissolves the antibiotic salt less. Examples of the latter are less polar, organic solvents, such as alcohols, acetone, acetonitrile and others. The mixture of water and methanol forms a special case. While these pure solvents readily dissolve A1437-D Ca salts, the mixtures of both solvents have distinctly poorer solvent properties. The A1437-D monocalcium salts (3d/iv) can thus be precipitated and crystallized from water (from methanol) with addition of methanol (water). In this way, calcium salts can be prepared or purified. The A1437-D monocalcium salt (3d/iv) readily forms gels in water-methanol mixtures. This gel formation is unfavorable for crystallization, since the crystallization rate is severely retarded thereby. For crystallization, measures must therefore be taken to suppress gel formation. Thus one measure can be, for example, the addition of small amounts of a suitable substance, such as, for example, butanol.
Another process for the preparation of the calcium salt of the compound of the formula II preferably of its monocalcium salt, for example the A1437-D monocalcium salt (3d/iv), consists in the use of a support, for example of adsorption resins, reverse-phase supports, molecular sieves and ion exchangers, which is loaded with an aqueous solution of a sodium or ammonium salt of the compound of the formula II (for example the sodium or ammonium salt of the compound A1437-D), which has been treated with a calcium halide, or alternatively with an aqueous solution of a dicalcium salt of the compound of the formula II (for example the dicalcium salts (3d/iia), (3d/iib) or (3d/iic)), after which the support is optionally washed with a suitable solvent and finally the calcium salt of the compound of the formula II, preferably the monocalcium salt, for example the A1437-D monocalcium salt (3d/iv), is eluted using a suitable solvent.
For use as adsorption resins, for example, Ambedite XAD 7 (Rohm and Haas), DIAION(copyright) HP20SS (Mitsubishi Chem. Corp.), Poros(copyright) 20 R2 or polyamide 6 (Riedel-deHaen) are suitable, when using reverse-phase supports, for example, LiChrosorb(copyright) RP-select B (E. Merck) is suitable. It is possible, however, also to employ supports which are usually used in hydrophobic interaction chromatography (HIC), for example for protein purification. Such supports are, for example, Phenyl Sepharose(copyright) or TSKgel Phenyl Toyopearl(copyright). Moreover, molecular sieves such as are used for xe2x80x9csize exclusion chromatographyxe2x80x9d or gel filtration chromatography are also suitable. The basis of this process is the tendency of the compound of the formula II (for example A1437-D) to bind calcium ions. When using the supports mentioned, a mixture of ammonium or sodium salts of the compound of the formula II with calcium salts is prepared in water and this mixture is separated on the supports in a manner known per se. Alternatively, it is also possible to apply an aqueous solution of an appropriate dicalcium salt. For example, an aqueous solution of A1437-D sodium salt and calcium chloride is applied to an adsorption resin, such as, for example, to DIAION HP(copyright) 20SS, the loaded resin is washed with water to remove excess salts, and then the calcium salt of the lipopeptide is eluted from the support using water-containing or anhydrous solvents, preferably using methanol. The A1437-calcium-containing fractions are dried. A product obtained in this way, for example, has the elemental composition C59H92N14O19Ca (monocalcium salt of the compound of the formula II (3d/iv)).
For obtaining calcium salts of the compound of the formula II, it is furthermore possible to employ ion exchangers, preferably anion exchangers. In this method, for example, any desired aqueous, low-ion solution of the compound of the formula II is bound to an anion exchanger at pHs between pH 5 and pH 9, and after washing the loaded support with water the monocalcium salt of the compound of the formula II is eluted using a rising concentration of a calcium salt which is soluble in water. The column eluate, which contains the monocalcium salt, is desalted, for example by reverse osmosis, and dried. Alternatively, the monocalcium salt can be isolated by other processes, such as, for example, by crystallization.
The compounds of the formula II, the precursors of the calcium salts according to the invention, can be prepared advantageously, as disclosed in EP 0 629 636 A1, by fermentation of Actinoplanes sp., preferably Actinoplanes friulensis (DSM 7358), and alternatively, as described in EP 0 688 789 A1 (U.S. Pat. No. 5,629,288), derivatized by replacement of the acid radical R1 in formula II by an acid radical which does not occur naturally. The compounds of the formula II thus obtained can be reacted as described above to give their calcium salts.
The object set at the outset, to make available an improved process for the fermentative preparation of the compound of the formula II, the acid precursor of the calcium salts according to the invention, in which preferably the compound of the formula II is produced by the microorganism, is achieved in that in the fermentation of Actinoplanes spec., preferably of Actinoplanes friulensis DSM 7358, the fermentation solution is supplemented with one or more complexing agents, preferably chelating agents, and with the amino acid asparagine.
Preferably, the complexing agent used is citric acid or ethylenediamine tetraacetate (EDTA).
Advantageously, it is also possible to supplement the fermentation solution with EDTA and citric acid.
To increase the yield of a compound of the formula II in which R, for example, is a fatty acid radical of the formula (3a) or preferably (3b), the fermentation solution can additionally be supplemented with the amino acid L-leucine. To increase the yield of a compound of the formula II in which R1, for example, is a fatty acid radical of the formula (3c) or preferably (3d), the fermentation solution can additionally be supplemented with the amino acid L-valine.
Accordingly, the present invention also relates to a process for the preparation of a calcium salt of the compound of the formula II, which is distinguished in that in a first step the compound of the formula II, as described above, is prepared by fermentation of Actinoplanes spec., preferably Actinoplanes friulensis (DSM 7358), the fermentation solution being supplemented with one or more complexing agents, preferably chelating agents, and with the amino acid asparagine, and in a subsequent step the calcium salt of the compound of the formula II, as explained above, is obtained by precipitation, crystallization or treatment on a support.
The present invention also relates to a calcium salt of.the compound of the formula II for use as a pharmaceutical.
The calcium salts of the compound of the formula II are preferably suitable for the production of a pharmaceutical against bacterial infections, the calcium salts (3c) and in particular (3d) or the dicalcium salts (3c/iia), (3c/iib) and (3c/iic) and in particular the dicalcium salts (3d/iia), (3d/iib) or (3d/iic) described above being particularly suitable, the dicalcium salt (3d/iia) being particularly preferred. The monocalcium salts of the compound of the formula II are particularly suitable for the preparation of a pharmaceutical against bacterial infections, the monocalcium salts (3c) and in particular (3d) having the empirical formula (3c, 3d/iv) C59H92N14O19Ca being preferred. The abovementioned calcium salts of the compound of the formula II are preferably suitable for the production of a pharmaceutical against bacterial infections which are caused by Gram-positive bacteria, preferably by glycopeptide-resistant bacteria.
The pharmaceuticals comprising at least one calcium salt of the compound of the formula II can furthermore comprise the customary pharmaceutical auxiliaries.
For example, the dicalcium salt of the compound of the formula II, preferably the chloride 3d/iia, can be dissolved in water containing equal parts by weight of mannitol and then lyophilized for the production of a pharmaceutical.
The calcium salts of the compound of the formula II are particularly suitable on account of their solubility and their toxicological properties for parenteral administration in the form of an injectable solution. Accordingly, the present invention also relates to injectable solutions, comprising one or more calcium salts of the compound of the formula II, preferably the calcium salt (3d/iv) having the empirical formula C59H92N14O19Ca or particularly preferably the calcium salt 3d/iia having the empirical formula C59H92N14O19Ca2Cl2.
For the production of an injection solution, the lyophilizate consisting of equal parts by weight of mannitol and calcium salt can be dissolved in suitably prepared water.